AU2021258029A1 - Application of a bile acid composite bacterial agent in the preparation of feed additives for mutton sheep - Google Patents
Application of a bile acid composite bacterial agent in the preparation of feed additives for mutton sheep Download PDFInfo
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- bile acid
- mutton sheep
- bacterial agent
- acid composite
- composite bacterial
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- 230000001580 bacterial effect Effects 0.000 title claims abstract description 64
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 title claims abstract description 55
- 239000003613 bile acid Substances 0.000 title claims abstract description 55
- 241001494479 Pecora Species 0.000 title claims abstract description 42
- 239000003795 chemical substances by application Substances 0.000 title claims abstract description 35
- 239000002131 composite material Substances 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 239000003674 animal food additive Substances 0.000 title claims abstract description 8
- 241000606210 Parabacteroides distasonis Species 0.000 claims abstract description 32
- 238000003307 slaughter Methods 0.000 claims abstract description 18
- 239000000725 suspension Substances 0.000 claims abstract description 17
- 238000000855 fermentation Methods 0.000 claims abstract description 3
- 230000004151 fermentation Effects 0.000 claims abstract description 3
- 229930006000 Sucrose Natural products 0.000 claims description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- DKPMWHFRUGMUKF-UHFFFAOYSA-N (3alpha,5alpha,6alpha,7alpha)-3,6,7-Trihydroxycholan-24-oic acid Natural products OC1C(O)C2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 DKPMWHFRUGMUKF-UHFFFAOYSA-N 0.000 claims description 3
- RUDATBOHQWOJDD-UHFFFAOYSA-N (3beta,5beta,7alpha)-3,7-Dihydroxycholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 RUDATBOHQWOJDD-UHFFFAOYSA-N 0.000 claims description 3
- JOYGXTIHTHBSOA-UHFFFAOYSA-N 1-(4-chlorophenyl)-3-thiophen-2-ylprop-2-en-1-one Chemical compound C1=CC(Cl)=CC=C1C(=O)C=CC1=CC=CS1 JOYGXTIHTHBSOA-UHFFFAOYSA-N 0.000 claims description 3
- DGABKXLVXPYZII-UHFFFAOYSA-N Hyodeoxycholic acid Natural products C1C(O)C2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 DGABKXLVXPYZII-UHFFFAOYSA-N 0.000 claims description 3
- RUDATBOHQWOJDD-BSWAIDMHSA-N chenodeoxycholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-BSWAIDMHSA-N 0.000 claims description 3
- 229960001091 chenodeoxycholic acid Drugs 0.000 claims description 3
- 230000003203 everyday effect Effects 0.000 claims description 3
- DGABKXLVXPYZII-SIBKNCMHSA-N hyodeoxycholic acid Chemical compound C([C@H]1[C@@H](O)C2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 DGABKXLVXPYZII-SIBKNCMHSA-N 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 230000003213 activating effect Effects 0.000 claims description 2
- 238000009395 breeding Methods 0.000 abstract description 6
- 230000001488 breeding effect Effects 0.000 abstract description 5
- 210000000577 adipose tissue Anatomy 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 235000016709 nutrition Nutrition 0.000 abstract description 3
- 230000035764 nutrition Effects 0.000 abstract description 3
- 238000010521 absorption reaction Methods 0.000 abstract description 2
- 238000000354 decomposition reaction Methods 0.000 abstract description 2
- 238000001727 in vivo Methods 0.000 abstract description 2
- 231100000331 toxic Toxicity 0.000 abstract description 2
- 230000002588 toxic effect Effects 0.000 abstract description 2
- 241000283707 Capra Species 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 239000002609 medium Substances 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 239000006150 trypticase soy agar Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- 238000007127 saponification reaction Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000003085 diluting agent Substances 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 239000008223 sterile water Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 210000003608 fece Anatomy 0.000 description 3
- 238000009629 microbiological culture Methods 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 210000000593 adipose tissue white Anatomy 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 238000004042 decolorization Methods 0.000 description 2
- 239000003925 fat Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000020477 pH reduction Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000021053 average weight gain Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000009304 pastoral farming Methods 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/105—Aliphatic or alicyclic compounds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/0216—Bacteriodetes, e.g. Bacteroides, Ornithobacter, Porphyromonas
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
- A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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Abstract
The present invention discloses application of a bile acid composite bacterial agent in the
preparation of feed additives for mutton sheep. The bile acid composite bacterial agent
comprises Parabacteroides distasonis bacterial suspension and bile acid, and the
Parabacteroides distasonis bacterial suspension is obtained by cultivation and fermentation of
Parabacteroides distasonis LCG-06 with the deposit number CGMCC No. 20820. The bile acid
composite bacterial agent of the present invention has natural components and has no toxic and
side effects, and can significantly increase the growth rate of mutton sheep and promote
nutrition absorption, accelerate the decomposition of in vivo fat of mutton sheep, thereby
reducing the body fat percentage of mutton sheep, increasing the slaughter weight and slaughter
rate of mutton sheep, increasing the incomes for breeding of mutton sheep. ThereforBIANGe,
it has broad application prospects.
Description
The present application claims the benefit of Chinese Patent Application No. 202011306541.9 filed on November 19, 2020, the contents of which are hereby incorporated by reference.
The present invention generally relates to the field of poultry and livestock breeding, and .o particularly to application of a bile acid composite bacterial agent in the preparation of feed additives for mutton sheep.
Mutton sheep is one of the most adaptable domestic animals to the external environment. With the improvement of the living standards of Chinese people, the demand for mutton sheep .5 has gradually increased. Presently, allopatry fattening way is mainly adopted for mutton sheep. There are many ways for crops and livestock breeding. In the pasturing areas, due to prohibition and restriction of grazing, the feeding amount of ewes is reduced, leading to shortage of sheep. Moreover, due to lack of feeding standard, some fattening farms have no scientific feed ratio, which causes different sizes of mutton sheep. When there is lack of nutrition, yellow fat disease or increased fat content may occur, which will affect the quality of mutton sheep and the farming incomes of farmers. In addition, some veterinary drugs or other feed additives are used, which may have side effects and have residue in the body of mutton sheep. When people eat such mutton sheep, their health will be seriously threatened. Therefore, it is very necessary to develop an efficient natural bacterial agent that can improve the quality of mutton sheep.
In order to solve the shortcomings of the prior art, the present invention provides application of a bile acid composite bacterial agent in the preparation of feed additives for mutton sheep. The bile acid composite bacterial agent can reduce body fat and slaughter weight of mutton sheep.
In order to achieve the object, the present invention adopts the following technical solutions:
The present invention provides a bile acid composite bacterial agent used as feed additives
for mutton sheep, wherein the bile acid composite bacterial agent comprises a
Parabacteroidesdistasonis bacterial suspension and bile acid.
Further, the Parabacteroidesdistasonis bacterial suspension is obtained by culture and fermentation of a Parabacteroides distasonis LCG-06 , wherein the Parabacteroides distasonis LCG-06 was deposited at China General Microbiological Culture Collection Center (CGMCC) with the deposit number CGMCC No. 20820.
Further, the bacterial count of the bile acid composite bacterial agent is not less than 5 x108 CFU/mL.
.0 Further, the method of using the bile acid composite bacterial agent is: adding the bile acid composite bacterial agent to feeds for mutton sheep to feed for 20 to 40 consecutive days at an amount of 30 to 50 mL/sheep/day.
Further, the bile acid composite bacterial agent is added to feeds for mutton sheep at noon every day to feed mutton sheep.
.5 Further, the bile acid composite bacterial agent can increase the slaughter weight and slaughter rate of mutton sheep.
A method for preparing a bile acid composite bacterial agent comprises the following steps:
(1) placing a Parabacteroidesdistasonis LCG-06 in a liquid nutrient medium and activating in an anaerobic environment at 37°C for 40 to 48 h; adding a sucrose solution with mass concentration of 20% to the activated bacteria solution, mixing well and centrifuging to precipitate, adding a sucrose solution with mass concentration of 10% to the precipitate, resuspending uniformly to obtain a bacterial suspension of Parabacteroidesdistasonis LCG 06;
wherein the Parabacteroidesdistasonis LCG-06 was deposited at China General Microbiological Culture Collection Center (CGMCC) with deposit number CGMCC No. 20820;
(2) mixing the bacterial suspension of Parabacteroidesdistasonis LCG-06 with the bile acid uniformly to obtain a bile acid composite bacterial agent.
Further, the volume-to-mass ratio of the bacterial suspension of Parabacteroides distasonis LCG-06 to the bile acid is 8-10:1.
Further, the bacterial count of the bacterial suspension of Parabacteroidesdistasonis LCG-06 is not less than 2x109 CFU/mL.
Further, the bile acid contains no less than 78% hyocholic acid and hyodeoxycholic acid and no less than 18% chenodeoxycholic acid.
Compared with the prior art, the present invention has the following advantages and beneficial effects. In the present invention, a strain Parabacteroidesdistasonis LCG-06 is screened from the feces in the intestinal tract of pigs, which will not cause harm to animal and human body, and a sucrose solution is used to prepare ParabacteroidesdistasonisLCG-06 into .0 a bacterial suspension, which is then compounded with bile acid in an optimized ratio to prepare a composite bacterial agent. The composite bacterial agent has natural components and has no toxic and side effects, and can significantly increase the growth rate of mutton sheep and promote nutrition absorption, accelerate the decomposition of in vivo fat of mutton sheep, thereby reducing the body fat percentage of mutton sheep, increasing the slaughter weight and .5 slaughter rate of mutton sheep, increasing the incomes for breeding of mutton sheep. Therefore, it has broad application prospects.
The sole figure shows bacterial colonies of Parabacteroidesdistasonis LCG-06 on the TSA medium.
The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. The experimental methods that do not indicate specific conditions in the following examples are usually in accordance with conventional conditions or conditions recommended by the manufacturers.
Example 1:
I. Screening and identification of Parabacteroidesdistasonis LCG-06
1. Screening of Parabacteroidesdistasonis LCG-06
Intestinal feces were taken from pig's intestines, 10 g of feces were mixed with 100 mL of sterile water, filtered to remove impurities and make a fecal diluent, and then sterile water was added to the diluent continuously, to make 1-1, 10-3, 10-4, 10- diluents. Then 10-2, 10-3, 10-4, 10-' diluents were spread on the Trypticase Soy Agar medium (TSA), and cultured at 37°C for 4 days in an anaerobic environment containing 80% N 2 , 10% CO2, and 10% H 2
. Obvious colonies were selected for multiple purifications to obtain a single colony, and named as LCG-06, and then placed in the TSA slant medium for refrigeration and storage. The formula of the TSA medium was as follows:
Tryptone 15.0g, soytone 5.0g, sodium chloride 5.0g, agar 15.0g, sterile water 1000 mL, pH 7.3+0.2.
As shown in the sole figure, the biological characteristics of the bacterial colonies of the .0 strain LCG-06 in TSA medium were as follows: circular or quasi-circular, milky white or off white, flat, 0.2 to 1.8 mm in diameter, smooth and dull surface, semitransparent, with neat edges.
2. Identification of Parabacteroidesdistasonis LCG-06
The genomic DNA of the strain LCG-06 was extracted and used as a template to perform .5 PCR amplification using 16S rRNA universal primers. The 16S rRNA amplified sequence was obtained and sequenced, and then the sequence Blast was performed. Results showed that the strain LCG-06 has the highest homology with Parabacteroidesdistasonisin the GenBank gene bank, so the strain LCG-06 was determined to be Parabacteroidesdistasonis.
The screened Parabacteroidesdistasonis LCG-06 was deposited in China General Microbiological Culture Collection Center (CGMCC) at Nio.3, 1 Beichen West Road, Chaoyang District, Beijing, Institute Of Microbiology Chinese Academy of Sciences; the deposit date: September 25, 2020; and the deposit No. ofParabacteroidesdistasonis:CGMCC No.20820.
3. Preparation of bacterial suspension of Parabacteroidesdistasonis LCG-06
(1) Pick the deposited Parabacteroidesdistasonis LCG-06 into a liquid nutrient medium (tryptone 15.0g, soytone 5.0g, sodium chloride 5.0g, sterile water 10OOmL, pH 7.4), activate and culture in an anaerobic environment at 37°C for 48h, to obtain bacterial solution of Parabacteroidesdistasonis LCG-06;
(2) Add 20% sucrose solution with a mass concentration to the bacterial solution at a volume ratio of 1:1, mix well, and centrifuge at 10,000 rpm/min for 5 min to obtain a bacterial strain precipitate;
(3) Re-dissolve the above bacterial strain precipitate with a sucrose solution with a mass concentration of 10% according to a ratio of the volume of the sucrose solution to the mass of the bacterial strain precipitate of 2:1, mix well to obtain the bacterial suspension of Parabacteroidesdistasonis LCG-06, with bacterial count of not less than 2x109 CFU/mL.
II. Preparation of bile acid
The process steps for preparing bile acid were as follows:
(1) Saponification: Add 1kg of porcine gall powder crushed through a 40-mesh sieve into a reactor, then add 1OL of sodium hydroxide solution with a mass concentration of 10%, heat and stir until boiling, and keep boiling and stir for 14h, then cool the saponification fluid until .0 solid-liquid stratification, then remove the supernatant, to obtain the remaining solid of bile acid saponification product;
(2) Decolorization: Add 6L of water to the bile acid saponification product, heat to 80°C to completely dissolve the bile acid saponification product, then pour it into a decolorization tank, add 300 mL of hydrogen peroxide and stir evenly, react at room temperature for 24 h, .5 then filter the reaction solution to an acidification tank, to obtain the filtrate;
(3) Acidification: Cool the filtrate to room temperature, slowly add a 10% hydrochloric acid solution, stir while adding, and stop adding acid when the pH of the solution reaches 3, and centrifuge and filter to obtain a white solid;
(4) Purification: Rinse the white solids with water continuously, to remove the water o soluble impurities attached to the surface of the white solids; after the filtered water is tested to be neutral, stop washing; place the white solids after centrifugal filtration into an oven, dry at 100°C until less than 10% moisture, to obtain the crude bile acid;
(5) Recrystallization: Place the crude bile acid into an extraction tank, add ethyl acetate of 10 times the weight of the crude bile acid to stir until dissolved completely, then add 0.08g of anhydrous sodium sulfate for dehydration, filter and collect the filtrate, concentrate the filtrate under reduced pressure and recover ethyl acetate to obtain liquid bile acid;
(6) Drying: Place the liquid bile acid into an oven and dry at 100°C until no more than 1% moisture, to obtain a bile acid finished product.
The bile acid finished product contains 78.6% of hyocholic acid and hyodeoxycholic acid and 20.0% of chenodeoxycholic acid.
III. A method for preparing a bile acid composite bacterial agent
The above prepared bile acid and bacterial suspension of Parabacteroidesdistasonis LCG-06 were mixed uniformly at a ratio of 1:10 (g/mL), to obtain a bile acid composite bacterial agent, with the total bacterial count of no less than 5x10 CFU/mL.
Example 2
Five hundred Boer goats were raised in a farm in Shandong, and 20 goats were randomly selected as the experimental group, and 20 goats as the control group, and all of 40 goats were of 4-5 generations. One month before the slaughter, the goats in the experimental group were fed with the feeds containing bile acid composite bacterial agent at noon every day, and the bile acid composite bacterial agent was evenly added to the feed at an additive amount of 30 to 50 mL per goat, and fed to goats continuously for 25 days. The goats in the control group were fed the same amount of feed, without adding bile acid composite bacterial agent. The feed was made of concentrated feed and grass feed in the same mixing ratio, and all mutton sheep continued to be raised in accordance with conventional breeding methods. 25 days later, goats were slaughtered, and the average slaughter weight of the mutton sheep in the control group was 56.44 kg, and more yellow and white fat was seen during slaughter; the average slaughter weight of the goats in the experimental group was 54.47 kg, with less yellow and white fat, and the mutton sheep in the experimental group had more weight gain during the 25-day breeding process, and higher average slaughter rate than that of the control group, indicating that the bile acid composite bacterial agent of the present invention can effectively reduce the body fat o percentage of the mutton sheep, and increase the slaughter weight and average slaughter rate of mutton sheep.
Average Initial average Average Weight gain slaughter slaughter Wegtan weight weight (kg) s(kg) rate weight (kg) (kg) (%)
Experimental 45.21 54.47 9.26 24.74 54.53 group
Control group 48.60 56.44 7.84 28.31 50.17
The above embodiments are only used to illustrate the technical solutions of the present invention rather than limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can make modifications to the technical solutions recorded in the foregoing embodiments or make equivalent replacement on some of the technical features; and these modifications or replacements shall not deviate from the spirit and scope of the technical solutions claimed by the present invention.
Claims (10)
1. Application of a bile acid composite bacterial agent in the preparation of feed additives for mutton sheep.
2. The application according to claim 1, wherein the bile acid composite bacterial agent comprises Parabacteroides distasonis bacterial suspension and bile acid.
3. The application according to claim 2, wherein the Parabacteroides distasonis bacterial suspension is obtained by culture and fermentation of Parabacteroides distasonis LCG-06 with the deposit number CGMCC No. 20820.
4. The application according to claim 2, wherein the bacterial count of the bile acid composite bacterial agent is not less than 5x108 CFU/mL.
5. The application according to claim 1, wherein the preparation of the bile acid composite bacterial agent comprises the following steps: (1) placing the Parabacteroides distasonis LCG-06 in a liquid nutrient medium and activating in an anaerobic environment at 37°C for 40 to 48 h; adding a sucrose solution with mass concentration of 20% to the activated bacteria solution, mixing well and centrifuging to precipitate, adding a sucrose solution with mass concentration of 10% to the precipitate, resuspending uniformly to obtain a bacterial suspension of Parabacteroides distasonis LCG 06; (2) Mixing the bacterial suspension of Parabacteroides distasonis LCG-06 with the bile acid uniformly to obtain a bile acid composite bacterial agent.
6. The application according to claim 5, wherein the volume-to-mass ratio of the bacterial suspension of Parabacteroides distasonis LCG-06 to the bile acid is 8-10:1.
7. The application according to claim 5, wherein the bile acid contains no less than 78% hyocholic acid and hyodeoxycholic acid and no less than 18% chenodeoxycholic acid.
8. The application according to claim 1, wherein the method of using the bile acid composite bacterial agent is: adding the bile acid composite bacterial agent to feeds for mutton sheep to feed for 20 to 40 consecutive days at an amount of 30 to 50 mL/sheep/day.
9. The application according to claim 8, wherein the bile acid composite bacterial agent is added to feeds for mutton sheep at noon every day to feed mutton sheep.
10. The application according to claim 1, wherein the bile acid composite bacterial agent can increase the slaughter weight and slaughter rate of mutton sheep.
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